The present invention relates to a method of measuring an angle of rotation usable for identifying, examining a purity and determining a concentration of a solute in the solution, and a polarimeter using the method, and more particularly to a method and an apparatus for urinalysis in which the angle of rotation of urine sampled from a man or other animal for examining the concentration of glucose, protein, etc. contained in the urine.
A healthy adult person usually voids 1000-1500 ml of urine every day. The total amount of solid components thereof is 50-70 g. About 25 g of the solid components is inorganic substances mainly composed of sodium chloride, potassium chloride and phosphoric acid, most of which are dissolved in the form of ions. The remains are organic substances mainly composed of urea and uric acid, and slight amounts of sugar and protein also exist therein. The concentrations of sugar and protein in the urine reflect the health conditions of the adult.
The sugar contained in the urine, i.e., glucose is discharged usually at a rate of 0.13-0.5 g per day into the urine. From this figure and the amount of urine, the concentration, i.e., the urine glucose level can be estimated at not more than 50 mg/dl on the average. The corresponding value is several hundred mg/dl, or sometimes as high as several thousand mg/dl. In other words, the value for diabetics can increase by a factor of ten or hundred as compared with the normal value.
On the other hand, the protein contained in urine, i.e., albumin is smaller in amount than glucose, and discharged at the rate of 3-60 mg into the urine. By taking the amount of the urine into account, average concentration is about 6 mg/dl or less. If a kidney is suffered, the albumin concentration reaches 100 mg/dl or more. That is, the value is increased to ten times the normal value or more.
Ordinally, as a conventional method of examining such sugar or protein in the urine, a test paper impregnated with an agent is dipped into the urine and a color reaction thereof is measured by spectrophotometer or the like.
In this method, however, different kinds of test paper were required to use for different items of examination including sugar, protein, etc. Also, a new test paper is required for each test, thereby leading to the disadvantage of a high running cost. Further, automation for labor saving has its own limit.
In addition, in a case of home use, a layman is demanded to set and change the test paper. This process is comparatively annoying and forms a stumbling block to the extension of the domestic use of the urinalysis apparatus.
Now, the conventional polarimeter will be explained. The conventional polarimeter had the problems described below.
An example of the conventional polarimeter is shown in FIG. 20.
In FIG. 20, a light source 121 is configured of a sodium lamp, a band-pass filter, a lens, a slit, etc. for projecting a substantially parallel light composed of a sodium D ray having a wavelength of 589 nm. A polarizer 122 is arranged in the direction of advance of the light projected from the light source 121 in such a position as to transmit only a component in a specific direction, which has a plane of vibration coincident with a transmission axis thereof, of the light projected from the light source 121. A sample cell 123 for holding a specimen is arranged in the direction of advance of the light transmitted through the polarizer 122. Further, an analyzer 124 is arranged, like the polarizer 122, in such a position as to transmit only the component of the light in a specific direction. An analyzer rotator 125 is for rotating the analyzer 124 on an axis parallel with the direction of advance of the light projected from the light source 121 under the control of a computer 127. A light sensor 126 is for detecting the light projected from the light source 121 and transmitted through the polarizer 122, the sample cell 123 and the analyzer 124. The computer 127 controls the analyzer rotator 125 while recording and analyzing a signal from the light sensor 126.
The principle of this conventional example will be explained with reference to FIG. 21. In the figure the abscissa represents the relative angle xcex8 formed between the plane of vibration of the light transmitted through the polarizer 122 and the plane of vibration of the light transmitted through the analyzer 126. Herein, xcex8 is assumed to take zero when the angle between these two planes of vibration reaches xcfx80/2, i.e., in the orthogonal nicol state. The ordinate represents an intensity I of the light that has reached the light sensor 126 based on an output signal of the light sensor 126. Herein, the solid line indicates the output signal in the case where the specimen exhibits no optical rotatory power. Under this condition, the relation between xcex8 and I is shown by equation (1) mentioned below. Herein, a transmission loss and a reference loss of the sample cell 123 and the analyzer 122 respectively are ignored.
I=Txc3x97Ioxc3x97(cos xcex8)2xe2x80x83xe2x80x83(1)
where,
T: transmittance of specimen
Io: intensity of light incident to specimen
As apparent from equation (1), I changes with a change of xcex8, i.e., with the rotation of the analyzer 126, so that an extinction point with a minimum I appears for each xcfx80.
In the case where the specimen has an optical rotatory power and its angle of rotation=xcex1, on the other hand, the light intensity is represented by dashed line in FIG. 21 and given by equation (2).
I=Txc3x97Ioxc3x97{cos(xcex8xe2x88x92xcex1)}2xe2x80x83xe2x80x83(2)
As seen from this, a specimen having an optical rotatory power, as compared with a specimen having no optical rotatory power, has the angle associated with the extinction point displaced by xcex1. The angle of rotation can be measured by finding the displacement xcex1 of the angle associated with the extinction point by the computer 127.
In this method, however, S/N ratio of the output signal of the light sensor 126 is comparatively inferior for lack of a modulated component and it is difficult to accurately determine the extinction point. As a result, a specimen with a small xcex1 cannot be measured with high accuracy.
For this reason, a polarimeter shown in FIG. 22 is also used in order to improve an accuracy of determining the extinction point. In FIG. 22, a light source 141 is configured of a sodium lamp, a band-pass filter, a lens, a slit, etc. for projecting a substantially parallel light of sodium D ray having a wavelength of 589 nm. A polarizer 142 and an analyzer 144 are arranged in the direction of advance of the light projected from the light source 141 aligning their transmission axes with the direction of advance of the light projected from the light source 141, with a sample cell holding a specimen interposed therebetween. An analyzer rotator 145 is for rotating the analyzer 144 on the transmission axis thereof as a rotation shaft under the control of a computer 147. A light sensor 146 detects the light projected from the light source 141 and transmitted through the polarizer 142, the sample cell 143 and the analyzer 144. The computer 147 controls the analyzer rotator 145, and records and analyzes the signal of the light sensor 146. An optical Faraday modulator 151 vibrates the direction of polarization. A signal generator 152 drives the optical Faraday modulator 151. A lock-in amplifier 143 is for phase sensitive detection of an output signal of the light sensor 146 with reference to the vibration-modulated signal from the optical Faraday modulator 151.
The operating principle of the polarimeter will be explained below with reference to FIG. 23.
In FIG. 23, the abscissa and the ordinate represent, as same in FIG. 21, xcex8 and I, respectively, with the extinction point and the neighborhood thereof shown in an enlarged view. The optical Faraday modulator 151 vibration-modulates the direction of polarization with an amplitude of xcex4 and an angular frequency of xcfx89. In the process, I is given as shown in equation (3) below from equation (2).
I=Txc3x97Ioxc3x97{cos(xcex8xe2x88x92xcex1+xcex4xc3x97sin(xcfx89xc3x97t))}2xe2x80x83xe2x80x83(3)
where
t: time
In FIG. 23, the extinction point or the neighborhood thereof is involved, i.e., xcex8≈xcfx80/2, and therefore equation (4) can be approximated as shown by equation (4).
xcex8≈xcfx80/2+xcex2xe2x80x83xe2x80x83(4)
where,
|xcex2| less than  less than 1
Substituting this equation (4) into equation (3) gives equation (5) below.
I=Txc3x97Ioxc3x97{[sin(xcex2xe2x88x92xcex1+xcex4xc3x97sin(xcfx89xc3x97t)]}2xe2x80x83xe2x80x83(5)
If it is assumed that an angle of rotation of the specimen and an amplitude of vibration modulation are small, that is |xcex1| less than  less than 1 and xcex4 less than  less than 1, equation (5) is approximated as shown in equation (6) below.
I≈Txc3x97Ioxc3x97{xcex2xe2x88x92xcex1+xcex4xc3x97sin
(xcfx89xc3x97t)}2=Txc3x97Ioxc3x97{(xcex2xe2x88x92xcex1)2+2xc3x97(xcex2xe2x88x92xcex1)xc3x97xcex4xc3x97sin
xcfx89xc3x97t)+[xcex4xc3x97sin(xcfx89xc3x97t)]2}=Txc3x97Io
{(xcex2xe2x88x92xcex1)2+2xc3x97(xcex2xe2x88x92xcex1)xc3x97xcex4xc3x97sin
(xcfx89xc3x97t)+[xcex42/2xc3x97(1xe2x88x92cos (2xc3x97xcfx89xc3x97t))]}(6)
This indicates that the output signal I of the light sensor contains signal components of angular frequency=0 (DC), xcfx89 and 2xc3x97xcfx89. This is obvious also from FIG. 15. By the phase sensitive detection of the value I with the vibration-modulated signal as a reference signal in the lock-in amplifier, it is possible to pick up the component of the angular frequency xcfx89, i.e., the value S shown in equation (7) below.
S=Txc3x97Io2xc3x97(xcex2xe2x88x92xcex1)xc3x97xcex4xe2x80x83xe2x80x83(7)
This S is zero only when xcex2=xcex1 and this point constitutes an extinction point. In other words, the value of xcex2 when S becomes zero in a step of rotating the analyzer and sweeping xcex2 is the angle xcex1 of rotation.
As described above, modulation of the direction of polarization, makes it possible to pick up the signal of the modulated frequency component selectively by separating it from noises such as a source light intensity, power fluctuations and radiation, thereby making it possible to obtain the signal S with high S/N. This value S can be used to determine the extinction point accurately and permits a highly accurate measurement of the angle a of rotation.
Nevertheless, the above-mentioned polarimeter is complicated in structure due to the need of a means for rotating the analyzer and a modulator, and therefore has its own limit of cost reduction and reliability.
Taking these subjects into consideration, the object of the present invention is to provide a method of urinalysis easy to maintain and manage without using supplies such as a test paper. Further, the object of the present invention is to provide a reliable, compact and inexpensive polarimeter and a urinalysis apparatus using this one.
According to the method of urinalysis of the present invention, a concentration of an optically active substance contained in urine is determined by measuring an angle of rotation of the urine. Glucose and protein existing in the urine exhibit an optical rotatory power whereas urea and uric acid constituting the main components of the organic substances in the urine have no optical rotatory power. Also, none of the inorganic substances in the urine exhibits the optically rotatory power. For this reason, the concentration of glucose or protein in the urine can be accurately determined by measuring the angle of rotation of the urine. Similarly, the concentration of L-ascorbic acid (what is called vitamin C) which may be contained in the urine can also be determined by measuring the angle of rotation. Once the angle of rotation of the urine is measured by using a high-precision polarimeter, therefore, the angle of rotation due to the optically active substances like glucose and protein existing in low concentration can be detected thereby making it possible to calculate the concentration of these substances. As a result, the concentration of the glucose and protein in the urine can be examined without using any supplies.
The present invention provides a highly accurate, reliable, compact and inexpensive polarimeter which solves the above-mentioned problems of the conventional polarimeter.
The principle of a method of analyzing the urine by measuring the angle of rotation according to the invention will be explained below.
The angle A of rotation is proportional to the product of a specific angle a of rotation and the concentration C of an optically active substance. This relation is shown by equations (8) and (9).
In the case where only one type of optically active substance is involved, the relation is given by
A [degree]=L[cm]xc3x97xcex1xc3x97C[kg/dl]xe2x80x83xe2x80x83(8)
while if N types of optically active substances are contained, the relation holds:
A=Lxc3x97(xcex11xc3x97C1+xcex12xc3x97C2 . . . +xcex1Nxc3x97CN)xe2x80x83xe2x80x83(9)
where L is the measured optical path length.
The specific angles a of rotation of glucose and albumin are shown in Table 1.
The specific angles of rotation shown above are values for a glucose aqueous solution and an albumin aqueous solution at 20xc2x0 C.
Specifically, when a light having a wavelength of 589 nm propagates by the distance of 10 cm through the glucose aqueous solution of 100 g/dl in concentration, a direction of polarization of the light rotates by 50 degrees. Although this concentration cannot be achieved due to the limited solubility of glucose actually, the direction of polarization rotates by 50xc3x9710xe2x88x923 degrees at the concentration of 100 mg/dl since the angle of rotation and the concentration are in proportion as shown in equation (8).
In the case where glucose is the only optically active substance in the urine, therefore, a urine glucose level can be calculated from the specific angle of rotation of glucose by measuring the angle of rotation of the urine. A similar calculation is possible also for albumin and L-ascorbic acid.
Further, an angle of rotation of urine having a known range of angle of rotation presented by an interfering optically active substance other than optically active substance of unknown concentration is measured, and the concentration C of the optically active substance is determined to be within the range of
(Axe2x88x92Ah)/(xcex1xc3x97L)xe2x89xa6Cxe2x89xa6(Axe2x88x92A1)/(xcex1xc3x97L)xe2x80x83xe2x80x83(10)
where A: measured angle of rotation of the urine [degree],
Ah: maximum value of the angle of rotation presented by the interfering optically active substance [degree],
A1: minimum value of the angle of rotation presented by interfering optically active substance [degree],
xcex1: specific angle of rotation of the optically active substance [degree/cm-dl/kg], and
L: measurement optical path length [cm].
According to this method, the concentration of each of a plurality of optically active substances including glucose, albumin, L-ascorbic acid, etc., which may coexist in urine can be calculated.
First, equation (9) is modified to obtain equation (11) below.
A=Ax+Adxe2x80x83xe2x80x83(11)
where it is assumed that
Ax=Lxc3x97xcex11xc3x97C1, and
Ad=Lxc3x97(xcex12xc3x97C2 + . . . +xcex1Nxc3x97CN).
In equation (2), assume that substance 1 is an optically active substance X to be detected, and substances 2-N are other optically active substances, i.e., interfering optically active substances, Ad corresponds to the angle of rotation presented by the interfering optically active substances. If the concentration range of the substances 2 to N is known, the maximum value Ah and the minimum value A1 that Ad can assume are known. This leads to equation (12) below.
Axe2x88x92Ahxe2x89xa6Ax=Axe2x88x92Adxe2x89xa6Axe2x88x92A1xe2x80x83xe2x80x83(12)
Equation (12) determines the range of the angle Ax of rotation, and the concentration Cx is also determined from the specific angle xcex1x of rotation and the length L of the measurement optical path. Equation (12) is expressed as equation (10) in terms of the concentration C of the optically active substance X.
In the case where the glucose concentration in urine is examined, for example, assume that the concentration of the interfering albumin is not more than 10 mg/dl, i.e., assume that the minimum value of the albumin concentration which can be assumed is 0 and the maximum value thereof is 10 mg/dl, respectively, with the wavelength of 589 nm and the length of measurement light path of 10 cm, Ah is zero degree and A1 is xe2x88x926xc3x9710xe2x88x923 degrees. Then, assuming that measurement A=0.1 degree, the glucose concentration C (mg/dl) can be determined to be 200xe2x89xa6Cxe2x89xa6212 from Table 1.
Actually, considering the fact that an abnormal urine glucose level can reach not less than several hundred mg/dl, the examination with the above-described accuracy often suffices. Specifically, in the case where the albumin concentration is about 10 mg/dl or less representing a normal value, the abnormality of the sugar in the urine can be determined by measuring the angle of rotation with a single wavelength.
Further, angles of rotation of the urine including N types of optically active substances is measured using the light having N different types of wavelength thereby to determine the concentrations of the optically active substances in the urine. The specific angle of rotation varies with wavelength due to optically rotatory dispersion. Consequently, in the case where N types of optically active substances coexist, N independent simultaneous equations can be obtained using equation (9) by measuring the angle of rotation by each of the N wavelengths. This makes it possible to calculate the concentration for N types of optically active substances.
In this way, the abnormality of the sugar or the albumin concentration in the urine can be determined by measuring the angle of rotation of the urine.
Also, the angle of rotation of the urine for the light having a wavelength of not less than 500 nm is measured. For the short wavelength of less than 500 nm, the absorption due to urochrome (a yellow component of urine), principally, increases to such an extent that the measurement accuracy is sometimes adversely affected.
Further, the concentration of a light-scattering substance contained in the urine is determined by measuring an amount of a light scattered in the urine.
Also, the light scattering substance is at least one of protein and blood. The molecular weight of the albumin constituting protein in the urine is about 70 thousands, which causes light scattering sufficiently large as compared with the light scattering due to the molecular weight of other organic substances including glucose (having a molecular weight of about 180) or the like and inorganic substances contained in the urine. Specifically, the scattering of the light propagating in the urine is dominated by albumin. As a result, the albumin concentration can be determined by irradiating light to the urine and observing the scattered light directly or in the form of the decreasing amount of the transmitted light. It is also possible to examine the presence or absence of the blood which have comparatively large particles.
Further, the amount of the scattered light in the urine is measured for the light having a wavelength of not less than 500 nm. As in the case of measuring the specific angle of rotation, the light having a wavelength of shorter than 500 nm involves an increased absorption mainly due to urochrome, often adversely affecting the measurement accuracy.
Furthermore, the amount of the scattered light is measured together with the angle of rotation of the urine thereby to determine the concentration of the light scattering substances as well as the optically active substances contained in the urine. This is effective especially in the case where both glucose and albumin exist in an amount not negligible in the urine.
In this way, according to the present invention, the concentration of sugar and protein in the urine can be easily and accurately determined by measuring the angle of rotation of the urine. This method also eliminates the need of supplies such as the test paper unlike in the prior method.
In addition, a high reliability and a low cost of the polarimeter is made possible, and a reduced cost and a higher reliability of the urinalysis apparatus is also made possible as described below.
According to the method of measuring the angle of rotation of the present invention, an angle of rotation of a specimen is measured by applying a magnetic field to the specimen and detecting the change in the direction of polarization of the light due to the magnetic field.
In view of this, the present invention provides a polarimeter comprising a monochromatic light source for projecting the light, a polarizer for transmitting only the polarized component in a specific direction of the projected light, a sample cell for holding the specimen and arranged in such a manner that the light passed through the polarizer is transmitted through the specimen, means for applying a magnetic field to the specimen, means for sweeping the magnetic field, an analyzer for transmitting only the polarized component in a specific direction of the light transmitted through the specimen, a light sensor for detecting the light transmitted through the analyzer, and calculation means for calculating an angle of rotation of the specimen on the basis of a magnetic field sweep signal of the magnetic field sweeping means and an output signal of the light sensor.
When a light is propagated through a medium and a magnetic field is applied in the direction of propagation of the light, the direction of polarization of the light is rotated in accordance with the propagation. This phenomenon is called the optical Faraday effect. This optical Faraday effect is given by equation (13) below.
a=Vxc3x97Hxc3x97Lxe2x80x83xe2x80x83(13)
where
a: rotational angle of the direction of polarization [minute],
V: Verdet""s constant of the medium [minute/A],
H: magnetic field [A/m], and
L: propagation distance [m].
The value V in equation (13) is varied with the medium, light wavelength and temperature. An example of V for various media is shown in Table 2.
This optical Faraday effect is utilized by the optical Faraday modulator used in the prior art. This is such that a solenoid coil is wound on a rod of flint glass and is supplied with a current to apply a magnetic field thereto, thereby modulating the direction of polarization of the light propagated in the direction of the magnetic field. Free modulation can be conducted by controlling the current flowing in the solenoid coil.
As described above, the optical Faraday effect permits the modulation of the polarization direction upon application of a magnetic field to a medium. As apparent from Table 2, this is also the case with water, chloroform, acetone or the like widely used as a solvent. Therefore, upon application of a magnetic field to a solution with a specimen dissolved therein, the very solution rotates the direction of polarization of the light propagating through the solution by the optical Faraday effect. Specifically, once a magnetic field is applied to a sample cell holding a specimen, the particular sample cell and the magnetic field application means function as an optical Faraday modulator. A solenoid coil, a magnet etc. which apply the magnetic field in the direction of light propagation can be used as a magnetic field application means. The magnetic field can be modulated by modulating the current flowing in the solenoid coil or by modulating the distance between the magnet and the specimen. In this way, the direction of polarization can be vibration-modulated by vibration-modulating the magnetic field, thereby making it possible to measure the angle of rotation in the same manner as in the prior art.
Also, if the magnetic field is swept, i.e., if the applied magnetic field is changed from a given strength to a different strength (including a change in polarity of the magnetic field), then the direction of polarization of the magnetic field can be rotated. By doing so, the same effect can be obtained as if the analyzer is rotated. Specifically, unlike in the prior art in which the displacement of the extinction point with the rotation of the analyzer is directly read from the angle of the analyzer, the method of measuring the angle of rotation according to the present invention is such that the displacement of the extinction point with the magnetic field swept is read by a current value, for example, which is converted into a magnetic field and further into an angle, thereby measuring the angle of rotation of the specimen. This is substantially the same as if a magnetic field is detected in which the angle of rotation generated by an optically active substance of a specimen coincides with the rotational angle of the direction of polarization due to the optical Faraday effect caused by an application of the magnetic field.
Sweeping of the magnetic field is not necessarily limited to a continuous change of strength but includes a discrete change thereof. In view of the fact that a change in the characteristic of an output signal of a light sensor with the rotation of the direction of polarization is generally known, the angle of rotation can be calculated by measurements taken at two or more points and interpolation or extrapolation from the measurements. Specifically, the angle of rotation of a specimen can be calculated from the output signals of the light sensor for at least two different magnetic fields. In such a case the measurement time can be shortened.